The amitochondrial parasitic protists, such as Entamoeba, Giardia, and Trichomonas, rely on glucose metabolism as their principal means of energy production. These protists, along with several other parasitic protozoans, have a form of phosphofructo-1-kinase(PFK) that utilizes inorganic pyrophosphate (PPi) as a phosphoryl donor. Because this form of PFK is not found in higher animals, PPi-dependent PFK represents an attractive target for the development of therapeutic agents that will interfere with vital energy metabolism in the parasite without affecting the host. The PPi-PFK represents not only a critical metabolic step in energy production, it acts also as a key glucogenic enzyme because the amitochondriate protozoans lack fructose bisphosphatase. Furthermore, it is a neutral pyrophosphatase, also absent in these organisms. The aims of the proposal are: (1) to improve the current expression system for Entamoeba histolytica PFK to allow the preparation of adequate quantities of homogeneous enzyme for analysis of structural and kinetic properties. (2) to examine structural correlates of catalysis of both Entamoeba and Naegleria PFKs utilizing site-directed mutagenesis. (3) to analyze the 3D structure of the Entamoeba PFK. Crystallization as a requisite for X- ray studies will be attempted as soon as over-expression is achieved. Prior to obtaining such information, computer graphic modeling will be used to derive 3 dimensional models of the PFK based upon the crystal structures of procaryotic PFKs and upon data from site-directed mutagenesis. (5) to employ a computer-driven docking program to search the Fine Chemicals Directory for molecules that will fit into the substrate binding site of Entamoeba PFK but not the binding site of mammalian PFK. Best fit molecules will be screened as inhibitors in enzyme assays. (6) to use axenically cultured Entamoeba to test the effectiveness of PFK inhibitors as inhibitors of protozoan growth.